Engineered proline hydroxylase biocatalysts for hydroxylation of chemical compounds

Abstract

The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

What is claimed is: 
     
       1. An engineered polypeptide having proline hydroxylase activity comprises an amino acid sequence having at least 90% sequence identity to reference sequence SEQ ID NO:604 and one or more residue differences as compared to SEQ ID NO:604 at residue positions selected from: 13, 14, 24, 26, 27, 30, 57, 61, 62, 72, 76, 77, 81, 82, 86, 88, 97, 114, 127, 128, 142, 158, 161, 163, 173, 175, 176, 178, 180, 184, 185, 186, 187, 188, 189, 191, 192, 195, 198, 200, 207, 209, 210, 211, 213, 215, 217, 218, 222, 225, 230, 233, 236, 238, 240, 241, 256, 259, 263, 265, 271, and 273. 
     
     
       2. The engineered polypeptide of  claim 1 , wherein said polypeptide has at least 90% sequence identity to SEQ ID NO: 604 or 640. 
     
     
       3. The engineered polypeptide of  claim 1 , wherein said polypeptide comprises residue differences at positions 26, 30, 62, 82, 114, 158, 161, 271, and 273, as compared to SEQ ID NO: 604. 
     
     
       4. The engineered polypeptide of  claim 1 , wherein said polypeptide comprises residue differences of 26A, 30N, 62D, 82K, 114S, 158N, 161P, 271W, and 273T, as compared to SEQ ID NO: 604. 
     
     
       5. The engineered polypeptide of  claim 1 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (25,55)-5-hydroxypipecolic acid. 
     
     
       6. The engineered polypeptide of  claim 5 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (25,55)-5-hydroxypipecolic acid with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold or more the activity of the naturally occurring enzyme. 
     
     
       7. The engineered polypeptide of  claim 5 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid with greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more diastereomeric excess of (2S,5R)-5-hydroxypipecolic acid. 
     
     
       8. A polynucleotide encoding the engineered polypeptide of  claim 1 . 
     
     
       9. A polynucleotide encoding the engineered polypeptide of  claim 3 . 
     
     
       10. The polynucleotide of  claim 8 , wherein said polynucleotide comprises a nucleic acid sequence optimized for expression in  E. coli.    
     
     
       11. The polynucleotide of  claim 9 , wherein said polynucleotide comprises a nucleic acid sequence optimized for expression in  E. coli.    
     
     
       12. An expression vector comprising the polynucleotide of  claim 8 , optionally further comprising at least one control sequence. 
     
     
       13. The expression vector of  claim 12 , wherein said vector comprises SEQ ID NO:1007, 1008, or 1009. 
     
     
       14. A host cell comprising the polynucleotide of  claim 8 . 
     
     
       15. A method of preparing an engineered polypeptide, comprising culturing the host cell of  claim 14 , under conditions suitable for expression of the polypeptide. 
     
     
       16. The method of  claim 15 , further comprising a step of isolating the engineered polypeptide.